Redox Biology 6 (2015) 552–564

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Redox Biology

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Research Paper Critical role of c-jun N-terminal kinase in promoting mitochondrial dysfunction and acute liver injury

Sehwan Jang a,1, Li-Rong Yu b,1, Mohamed A. Abdelmegeed a, Yuan Gao b, Atrayee Banerjee a, Byoung-Joon Song a,n a Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD 20892-9410, USA b Biomarkers and Alternative Models Branch, Division of Systems Biology, National Center for Toxicological Research, Food and Drug Administration, Jef- ferson, AR 72079, USA article info abstract

Article history: The mechanism by which c-Jun N-terminal protein kinase (JNK) promotes tissue injury is poorly un- Received 23 September 2015 derstood. Thus we aimed at studying the roles of JNK and its phospho-target in mouse models of Accepted 29 September 2015 acute liver injury. Young male mice were exposed to a single dose of CCl4 (50 mg/kg, IP) and euthanized Available online 9 October 2015 at different time points. Liver histology, blood alanine aminotransferase, and other activities fi Keywords: were measured in CCl4-exposed mice without or with the highly-speci c JNK inhibitors. Phosphoproteins Acute liver injury were purified from control or CCl4-exposed mice and analyzed by differential mass-spectrometry fol- Carbon tetrachloride lowed by further characterizations of immunoprecipitation and activity measurements. JNK was acti-

JNK vated within 1 h while liver damage was maximal at 24 h post-CCl4 injection. Markedly increased Protein phosphorylation phosphorylation of many mitochondrial proteins was observed between 1 and 8 h following CCl4 ex- Mitochondria posure. Pretreatment with the selective JNK inhibitor SU3327 or the mitochondria-targeted antioxidant Differential proteomics mito-TEMPO markedly reduced the levels of p-JNK, mitochondrial phosphoproteins and liver damage in

CCl4-exposed mice. Differential proteomic analysis identified many phosphorylated mitochondrial pro- teins involved in anti-oxidant defense, electron transfer, energy supply, fatty acid oxidation, etc. Alde- hyde dehydrogenase, NADH-ubiquinone , and α-ketoglutarate dehydrogenase were

phosphorylated in CCl4-exposed mice but dephosphorylated after SU3327 pretreatment. Consistently,

the suppressed activities of these were restored by SU3327 pretreatment in CCl4-exposed mice. These data provide a novel mechanism by which JNK, rapidly activated by CCl4, promotes mitochondrial dysfunction and acute hepatotoxicity through robust phosphorylation of numerous mitochondrial pro- teins. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. . Introduction toxic compounds such as acetaminophen (APAP), cocaine, and binge alcohol (ethanol). For instance, over 56,000 emergency room visits Epidemiological studies revealed that many people suffer from with more than 400 deaths per year are due to APAP-induced acute acute liver failure, which can be produced by overdose of potentially liver injury in the United States alone [1]. Over-exposure to these substances can cause cell/tissue injury and sudden deaths in hu- mans and experimental models [1,2 and references within] espe- cially in combination with alcohol through additive or synergistic Abbreviations: ALT, alanine aminotransferase; APAP, acetaminophen; CYP2E1, cytochrome P450 2E1; ERK, extracellular signal-regulated kinase; α-KGDH, α-ke- interactions [3,4]. However, the molecular mechanisms of acute li- toglutarate dehydrogenase; HAE, 4-hydroxyalkenal; JNK, c-Jun N-terminal protein ver damage by these agents remain elusive because of the con- kinase; MDA, malondialdehyde; MPT, mitochondrial permeability transition; flicting results. NdufS1, 75 kDa-subunit NAD þ -dependent ubiquinone-oxidoreductase; p38K, p38 α Carbon tetrachloride (CCl4) is a hepatotoxic solvent widely used kinase; PBS, phosphate buffered saline; PDH, pyruvate dehydrogenase; TNF- , tu- fi mor necrosis factor-alpha; WT, wild-type to study the mechanisms for acute liver injury and chronic brosis n Corresponding author. Fax: þ1 301 594 3113. in experimental models [5–8]. The majority of CCl4 is metabolized E-mail addresses: [email protected] (S. Jang), by the ethanol-inducible CYP2E1 and thus Cyp2e1-null mice are [email protected] (L.-R. Yu), protected from CCl4-induced liver damage [9]. Following CYP2E1- [email protected] (M.A. Abdelmegeed), related metabolism, free radical metabolites of CCl attack cellular [email protected] (Y. Gao), [email protected] (A. Banerjee), 4 [email protected] (B.-J. Song). membranes, producing lipid peroxides, leading to severe necrosis 1 These authors equally contribute to this manuscript. in the pericentral regions of the liver [5,6]. However, despite the http://dx.doi.org/10.1016/j.redox.2015.09.040 2213-2317/Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). S. Jang et al. / Redox Biology 6 (2015) 552–564 553 well-established role of lipid peroxidation in this model [5,6],itis (Ndufs1) of NADH-ubiquinone oxidoreductase (complex I), cyto- virtually unknown whether CCl4 can promote liver injury through chrome c oxidase (complex IV), ATP synthase beta-subunit (ATP5B, modifying the phosphorylation of cellular proteins. complex V), lamin, α-tubulin, peroxiredoxin, and α-ketoglutarate Mitogen-activated protein kinases (MAPKs) are cell signal-re- dehydrogenase (α-KGDH) were obtained from Santa Cruz Bio- lated kinases known to regulate cell death and growth, depending technology, Inc (Santa Cruz, CA, USA). Specific antibodies to 3-ni- on the cellular contexts and their temporal fluctuations. It is well- tro-Tyr (3-NT) were from Abcam (Cambridge, MA) while specific established that there are three major MAPKs: c-Jun N-terminal anti-HNE-adducts were purchased from Calbiochem (La Jolla, CA, protein kinase (JNK), p38 kinase (p38K), and extracellular signal- USA). Specific JNK inhibitors SU3327 [20] and BI-78D3 [21] were regulated kinase (ERK). In general, activation of JNK and p38K is purchased from Tocris Bioscience (Bristol, United Kingdom). Both related to cell death signaling pathways while ERK activation is SU3327 and BI-78D3 inhibit JNK activity through blockade of involved in cell survival and proliferation [10]. JNK-related sig- protein-protein interaction between JNK and its scaffolding pro- naling pathways are also known to be associated with liver phy- tein JNK-interacting protein-1 (JIP-1). In addition, another report siology and pathology [11]. For instance, JNK-mediated cell da- suggested that BI-78D3 can inhibit JNK activity through covalent mage has been observed with many toxic compounds such as modification of Cys163 of JNK [22]. Other agents including the JNK APAP [12–14], staurosporine [15] and UV exposure [15] as well as inhibitor SP600125 and the mitochondria-targeted antioxidant under pathological conditions including ischemia-reperfusion in- mito-TEMPO [2-(2,2,6,6-tetramethylpiperidine-1-oxyl-4-ylamino) jury [16]. Mendelson et al. [17] reported that JNK was potently 2-oxoethyl]tripphenylphosphenium chloride [23] were obtained activated while ERK and p38K remained unchanged and de- from Sigma (St. Louis, MO, USA). creased, respectively, following CCl4 treatment, suggesting a role of JNK in promoting hepatotoxicity. In contrast, other investigators 2.2. Animal treatment and histological analysis reported that JNK activation was not critical in CCl4-mediated acute liver injury, compared to the important role of JNK in APAP- Age and gender-matched young male NCI-WT mice (Svj-129 induced liver damage [12,13]. Therefore, the underlying mechan- background) and Cyp2e1-null mice [24], kindly provided by Dr. ism by which activated (phosphorylated) p-JNK promotes cell or Frank J. Gonzalez (National Cancer Institute, Bethesda, MD), were liver damage is poorly understood. used and kept in a 12 h light-dark cycle with food and water ad

We recently reported that CCl4 rapidly activated p-JNK, which libitum in accordance with NIH guidelines. After a single in- was translocated to mitochondria and phosphorylated mitochon- traperitoneal injection of CCl4 (50 mg/kg as 0.5% in corn oil), the drial aldehyde dehydrogenase (ALDH2), an anti-oxidant enzyme, WT and Cyp2e1-null mice (n¼4–5/group) were euthanized at the leading to its inactivation and accumulation of lipid peroxides indicated time points. Each JNK inhibitor (10 mg/kg, a single ip following CCl4 exposure [18]. In addition, a previous report with injection, n¼4–5/group) was prepared as 1.25 mg/mL with 10% isolated mitochondrial proteins showed that activated p-JNK Solutol-HS15 in PBS, and administered 30 min prior to CCl4 in- phosphorylated other mitochondrial enzymes such as pyruvate jection. Mito-TEMPO was dissolved in sterile saline and adminis- dehydrogenase (PDH) E1α and β subunits, ATP synthase α and β tered by a single dose at 5 mg/kg ip 30 min before CCl4 treatment. subunits, and heat shock proteins (Hsp60 and Hsp70) [19]. Based A portion from the largest lobe of each liver, obtained from on these reports, we hypothesized that p-JNK, rapidly activated at CCl4-exposed WT in the absence or presence of JNK inhibitors and early hours after CCl4 exposure, translocates to mitochondria, Cyp2e1-null mice collected at different time points, was fixed in phosphorylates many mitochondrial proteins and suppresses their 10% neutral buffered formalin. After paraffin embedding and the functions, contributing to mitochondrial dysfunction and necrotic cutting of 4 mm slices, all sections were stained with hematoxylin liver damage. However, it is poorly understood how many other and eosin (H&E), as described [24]. Histological evaluation was proteins are phosphorylated by p-JNK and whether these phos- performed in a blinded manner. phoproteins are functionally altered to contribute to CCl4-induced liver injury. Thus, we investigated the mechanism of acute hepa- 2.3. Mass spectrometry analysis of purified phosphorylated proteins totoxicity by determining the role of JNK in promoting mi- tochondrial dysfunction and liver necrosis. For this purpose, we To affinity-purify phosphorylated proteins, 250 mg of mi- specifically aimed to identify and characterize mitochondrial JNK- tochondrial proteins from WT mice at 2 h post-injection of CCl4, target proteins in the absence or presence of the specific JNK in- were subjected to a metal-affinity column (Qiagen, Valencia, CA) hibitors. To further support the role of JNK in causing hepato- by following the manufacturer’s instructions. The purification toxicity, we also evaluated the histological and biochemical mea- procedures for vehicle-control (control) and CCl4-exposed sam- surements of CCl4-exposed Cyp2e1-null mice as compared to those ples, respectively, were repeated at least five times to collect suf- of the corresponding wild-type (WT) counterparts. ficient amounts of purified phosphoproteins. Purified phospho- proteins were resolved on 1-D SDS-PAGE. Each gel lane was cut with a razor blade into 13 bands, transferred into clean tubes and 2. Materials and methods subjected to further digestion with sequencing grade trypsin (Promega, Madison, WI, USA). In-gel digestion of protein gel slices, 2.1. Chemicals and other materials nanoflow reversed-phase liquid chromatography (nanoRPLC)– tandem mass spectrometry (MS/MS) and protein identification NAD þ and propionyl aldehyde were purchased from Sigma (St. analyses were performed as recently described [25]. For mass Louis, MO, USA). Specific antibodies to c-Jun, phospho-c-Jun spectrometry analysis, each sample was re-dissolved in 15 μLof (Ser63), JNK, phospho-JNK (Thr183/Tyr185), p38 kinase, phospho- 0.1% formic acid and 5 μL was injected onto a 9 cm 75 μm i.d. in- p38 kinase (Thr180/Tyr182), ERK, phospho-ERK (Thr202/Tyr204), house packed fused silica capillary electrospray ionization (ESI) phospho-Ser-Pro, Bax, and cytochrome C were purchased from C18 column, which was coupled online to a liner ion trap mass Cell Signaling Technology, Inc (Beverly, MA). Phosphoprotein- spectrometer (LTQ XL, Thermo Electron, San Jose, CA). Peptide purification kit including the metal-affinity columns was obtained separation was performed at a flow rate of 250 nL/min using a from Qiagen (Valencia, CA). ProQ-Diamond phosphoprotein step gradient of 2–42% solvent B (0.1% formic acid in acetonitrile) staining reagent was from Invitrogen (Eugene, OR). Specific goat for 40 min, 42–98% solvent B for 10 min, and 98% solvent B for polyclonal antibodies to mitochondrial ALDH2, 75-kDa subunit 5 min. Both solvents A (0.1% formic acid in water) and B were 554 S. Jang et al. / Redox Biology 6 (2015) 552–564 delivered by an Agilent 1200 nanoflow LC system (Agilent Tech- measured by quantifying the production of NADH at 340 nm, as nologies, Palo Alto, CA). The mass spectrometer was operated in a previously described [31]. One unit of activity was defined as in- data dependent mode in which each full MS scan was followed by crease in the absorbance at 340 nm per 0.1 mg protein/min mul- 7 MS/MS scans where the 7 most abundant peptide molecular ions tiplied by 1000. NADþ -dependent ubiquinone-oxidoreductase were dynamically selected from the prior MS scan for collision- (mitochondrial complex I) activity was measured by the method induced dissociation (CID) using a normalized collision energy of previously described [25,32] except that rotenone-containing 35%. The raw MS/MS data were searched using the SEQUEST samples were measured in parallel. Rotenone-sensitive activities cluster running under BioWorks (Rev. 3.3.1 SP1) (Thermo Electron, were normalized with control sample. Activities of α-ketoglutarate San Jose, CA) against a mouse IPI proteome database downloaded dehydrogenase (α-KGDH) were determined as previously de- from the European Bioinformatics Institute (EBI) (http://www.ebi. scribed [33] with a modification where the reaction volumes were ac.uk) for the identification of peptides and proteins within each reduced to 0.2 mL and 0.1 mg mitochondrial proteins were used gel band. To identify phosphoproteins from CCl4-exposed samples, for each measurement. One unit of activity was defined as increase only the proteins with two or more unique peptides were con- in the absorbance at 340 nm per 0.1 mg protein/min multiplied by sidered confidently as legitimate identifications. Subtraction of 1000. Mitochondrial swelling index was determined as described phosphoproteins between control and CCl4-exposed samples was [34]. All activities were measured with a Synergy 2 microplate performed to further identify the proteins preferentially phos- reader (BioTek, Winooski, VT, USA). phorylated by CCl4 exposure [26] using the similar method de- scribed previously [27]. For this analysis, the area of extracted ion 2.6. Immunoprecipitation and immunoblot analysis chromatogram of each identified peptide was calculated. The total area of all the peptides identified from the same protein was then Immunoprecipitations of ALDH2, α-KGDH, and NdufS1 (the 75- normalized by the total area of all the proteins identified within kDa subunit of complex I) with the specific antibody to each the sample (13 gel bands for each sample) and expressed as parts protein were conducted by following the recommended protocol per million (ppm) that referred to normalized intensity. After of Dynabeads (Invitrogen-Life Technologies, Grand Island, NY, normalization, a ratio was calculated for each protein between the USA). A separate aliquot of mitochondrial proteins (0.5 mg each)

CCl4-exposed and the control samples. A ratio of CCl4/controlZ1.5 was incubated with 2 mg of antibody-conjugated beads overnight. was used to identify phosphoproteins following CCl4-exposure, The immunoprecipitated proteins were dissolved in Laemmli since CCl4 treatment markedly increased phosphorylation of mi- buffer for immunoblot analysis using the specific antibody against tochondrial proteins compared to those of control. The quantified each target protein, as indicated [29]. mitochondrial phosphoproteins were further analyzed using Me- taCore (GeneGo, St. Joseph, MI, USA) software for molecular toxi- 2.7. Intensity data processing and statistical analysis city analysis according to the method described previously [28] and for verification of mitochondrial localization of proteins in The intensity of immuno-recognized protein bands such as conjunction with UniProt Knowledgebase (http://www.uniprot. ALDH2, NdufS1 75-kDa subunit of mitochondrial complex I, α- org). KGDH, etc on 1-D gels was determined by using a gel digitizing software (UN-SCAN-IT™, Orem, Utah, USA), as previously de- 2.4. SDS-PAGE and immunoblot analysis scribed [18,25]. All data in this report represent the results from at least three separate experiments, unless stated otherwise. Statis- Mitochondrial fractions were prepared from pooled mouse li- tical analyses were performed using the Student's t test and vers (n¼4–5/group) from the different treatment groups, as pre- po0.05 was considered statistically significant. Other methods viously described [29,30]. CHAPS-solubilized mitochondrial pro- and materials not described were the same as reported previously teins were dissolved in Laemmli SDS-sample buffer and electro- [25,28–30] or described in the Supplemental files. phoresed as per manufacturer's recommendations (Bio-Rad, Her- cules, CA) and subjected to immunoblot analysis with the specific antibody to JNK, p-JNK, p-c-Jun, p-Ser-Pro, α-KGDH, ATP5B, 3. Results NdufS1, or ALDH2, as indicated. The images were visualized by enhanced chemiluminescence detection by following the manu- 3.1. Time-dependent JNK activation and its role in CCl4-induced liver facturer’s recommendations (Pierce, Rockford, IL, USA). injury

2.5. Measurements of liver injury parameters, enzyme activities, and Our results showed that serum ALT levels remained unchanged mitochondrial swelling index until 8 h but markedly increased at 24 h post-CCl4 injection (Fig. 1A). Consistently, liver histology showed severe pericentral Serum alanine aminotransferase (ALT) level from each mouse necrosis observed only at 24 h with little liver injury until 8 h was determined by using the automated IDEXX Catachem chem- post-injection (Fig. 1B). CCl4-induced liver damage is related to istry analyzer system (IDEXX Laboratories, West Brook, ME, USA). increased lipid peroxidation [5,6] and inflammation as reflected by The concentration of lipid peroxides as malondialdehyde (MDA)þ elevated levels of TNFα [13,35]. However, our data revealed that 4-hydroxyalkenal (HAE) (mM) was measured with a commercially the levels of lipid peroxide measured by MDAþHAE (Fig. 1C) and available kit (Oxford Biomedical Research, Oxford, MI, USA) by serum TNFα (Fig. 1D) remained unchanged up to 8 h, although following the manufacturer’s protocol. Serum TNFα-levels were they were significantly elevated at 24 h post-injection. determined by using an ELISA assay kit using the manufacturer’s Based on rapid JNK activation in CCl4-exposed rats [17,18,36], protocol (eBioscience, San Diego, CA). Anti-phospho-c-Jun (Ser63) we studied the temporal activation of MAPKs in CCl4-exposed antibody was used to measure JNK activity by immunoblot ana- mice by immunoblot analysis. JNK was not activated in corn-oil lysis. Mitochondrial and nuclear fractions from each group were (control) group, but potently activated (phosphorylated) at 1 and further prepared by subtractive centrifugation, as described 2 h post-CCl4 injection (Fig. 2A). Activated p-p38K was detected in [29,30]. The respective marker proteins for cytosol, mitochondria control and its levels did not change (i.e., decreased) until 4 h upon and nuclear fractions were shown to verify relative purity of each CCl4 exposure. In contrast, the activated p-ERK was detected in fraction (Supplemental Fig. S1). Mitochondrial ALDH2 activity was control while their levels were increased at 1 or 2 h and returned S. Jang et al. / Redox Biology 6 (2015) 552–564 555

50000 * 40000

30000

20000 ALT (U/L) ALT 10000

0 C1 2 824 Time (h)

12 * 40 * 10 35 8 30 (pg/mL) 25 6 20 4 15 10

MDA + HAE (µM) MDA 2 5

0 Serum TNF 0 C1 2 4 824 C1 2 4 824 Time (h) Time (h)

Fig. 1. Time-dependent JNK activation and its role in CCl4-induced hepatotoxicity in WT mice. (A) Time-dependent changes in the serum ALT levels and (B) typical H&E- stained liver slides are presented for each indicated group (magnification 100 ). Severe necrotic regions of CCl4-exposed samples are marked with broken lines in (B).

(C) Hepatic levels of MDAþHAE, and (D) serum TNF-alpha levels following CCl4 exposure are presented. *, Significantly different (po0.05) from the other groups.

Fig. 2. Time-dependent changes in JNK, phosphorylated mitochondrial proteins and JNK activation in CCl4–exposed WT or Cyp2e1-null mice. (A) Whole cell lysates (50 mg/ assay) for indicated samples from WT mice were subjected to immunoblot analysis with the specific anti-p-JNK, anti-p-p38K, anti-p-ERK, their respective non-phospho- proteins, anti-c-Jun, or anti-phospho-c-Jun antibody. (B) Mitochondrial lysates (50 mg/lane) for indicated samples from WT mice were subjected to immunoblot analysis using anti-p-Ser-Pro antibody (left) or anti-p-Thr-Pro antibody (right). Time-dependent changes in the serum ALT levels (C) and JNK activation (D) are presented. Whole cell lysates (50 mg/assay) for indicated samples (n¼4/each time point) were used to determine JNK activation by immunoblot analysis by using anti-p-JNK antibody (top) and anti-JNK antibody (bottom). 556 S. Jang et al. / Redox Biology 6 (2015) 552–564

to basal levels at 4 h post-injection (Fig. 2A). The activated 3.3. Protection of CCl4-induced liver injury by new selective JNK phospho-c-Jun was also detected between 2 and 8 h after inhibitors

CCl4-injection, while c-Jun levels were increased between 1 and 8h. To further determine the critical role of JNK in mitochondrial dysfunction and liver injury, we evaluated the effects of known chemical inhibitors of JNK on CCl -induced JNK activation and 3.2. JNK target proteins and role of CYP2E1 in CCl -induced JNK 4 4 translocation of p-JNK to mitochondria at 2 h and hepatotoxicity at activation 24 h post-CCl4 injection (n¼4–5/group). Pretreatment with ATP- competitive JNK inhibitor SP600125 did not prevent CCl -induced The levels of total mitochondrial phosphoproteins, detected by 4 liver injury determined by serum ALT levels and liver histology anti-p-Ser-Pro antibody, were markedly increased at 1 h and las- (Supplemental Fig. S2A and B, respectively), unlike the earlier re- ted up to 8 h post-CCl injection (Fig. 2B, left). However, the levels 4 ports [12,37]. However, pretreatment with a new specific JNK in- of mitochondrial phosphoproteins, detected by anti-p-Thr-Pro hibitor SU3327 [20] significantly protected against CCl4-induced antibody, were very similar between control and CCl -exposed 4 acute hepatotoxicity assessed by ALT levels (Fig. 3A) and liver samples, except for a few protein bands (Fig. 2B, right). These re- histology (Fig. 3B). Pretreatment with another specific JNK in- sults suggest that the number and levels of many phosphorylated hibitor BI-78D3 [21,22] also showed similar results of protection mitochondrial proteins were increased following CCl4 exposure, against CCl4-induced hepatotoxicity based on the serum ALT levels although some phosphoproteins existed in control mice. (Supplemental Fig. S2C). We also determined the levels of JNK activation at 2 h and the We further studied the effect of SU3327 on JNK activation and degrees of acute hepatotoxicity assessed by ALT and histology at translocation of p-JNK to mitochondria at 2 h post-CCl4 injection. JNK 24 h in CCl4-exposed WT and Cyp2e1-null mice. Similar to the was potently activated in CCl4-exposed WT mice (Fig. 3C, top panel, earlier reports [9], ALT levels (Fig. 2C) and histological liver da- second lane) compared to the control (first lane). SU3327 pretreat- mage (data not shown) were very low in CCl -exposed Cyp2e1-null 4 ment markedly reduced the intensity of active p-JNK in CCl4-exposed mice even at 24 h, compared to the corresponding WT mice mice (third lane). Consistently, SU3327 pretreatment markedly de- (Fig. 1A and B, respectively). In addition, the JNK was not activated creased the amount of active p-JNK translocated to mitochondria in Cyp2e1-null mice at any time points following CCl4 exposure, (Fig. 3D, top panel, third lane) compared with the corresponding compared to the corresponding WT mice (Fig. 2D). These results CCl4-exposed mice (second lane). In contrast, the levels of p-ERK in suggest that CYP2E1-mediated CCl4 metabolism is required for JNK cytoplasm and mitochondria were unchanged regardless of SU3327 activation, which becomes very important in causing acute hepa- pretreatment (Fig. 3C and D, middle panels, respectively). Conse- totoxicity usually observed at later time points [11–14,18]. quently, the levels of phosphorylated mitochondrial proteins causally

* 25000 Control 20000 15000 10000 CCl4 ALT (U/L) ALT 5000 0 CCCl4CCL4 + CCl4 + SU3327 SU3327

Cytosol Mitochondria Mitochondria

kDa kDa kDa 54 P-JNK 54 46 46 P-JNK 250 54 110 46 JNK 40 P-ERK IB: anti- 50 35 P-Ser-Pro 43 P-ERK 56 ALDH2 15 Prx 25 56 ATP5B 56 IB: anti-ATP5B

Fig. 3. Pharmacological inhibition of JNK activation alleviates the CCl4-induced liver injury. (A) Serum ALT levels and (B) H&E-stained liver histology (magnification 100 )at

24 h following CCl4 exposure in WT mice with or without SU3327 pretreatment are presented. *, Significantly different (po0.05) between the two groups. Severe necrotic regions are marked with broken lines. (C) Immunoblot analyses demonstrating relative JNK or ERK activation in the indicated groups treated with CCl4 for 2 h are presented. Peroxiredoxin (Prx, bottom panel) levels were presented as a loading control. (D) Mitochondrial proteins treated for 2 h (50 mg/lane) were subjected to immunoblot analysis with the specific antibody to phospho-JNK (top), p-ERK (second panel), ALDH2 (third panel), or ATP synthase β-subunit (ATP5B, bottom) used as a loading control.

(E) Relative levels of phosphorylated mitochondrial proteins in the indicated samples treated with CCl4 for 2 h are shown after determining their levels by immunoblot analysis by using anti-p-Ser-Pro antibody (top) or anti-ATP5B as a loading control (bottom). S. Jang et al. / Redox Biology 6 (2015) 552–564 557

15

* * 10

5 MDA+HAE (μM)

0 C CCl4 CCl4 + SU3327 24 h CCl4+ Control CCl4 mito-TEMPO CCl4+ Control CCl4 mito-TEMPO P-JNK

JNK P-Ser-Pro

ATP5B

2 h post-CCl4-injection

Fig. 4. Effects of SU3327 and mito-TEMPO on the levels of lipid peroxides, p-JNK and mitochondrial protein phosphorylation in CCl4-exposed mice. (A) The levels of lipid peroxides [MDAþHAE] at 24 h post-CCl4 injection without or with SU3327 pretreatment are shown. *, Significantly different (po0.05) between the two groups, as indicated.

The Immunoblot results for the levels of (B) active p-JNK and (C) mitochondrial phosphoproteins at 2 h following CCl4 exposure in WT mice without or with mito-TEMPO pretreatment are presented. Other parameters, including ATP synthase-β subunit (ATP5B, bottom) used as a loading control, are same as the Fig. 3 legend.

correlated with the levels of active p-JNK in CCl4-exposed mice in the greater than 1.5-fold revealed that at least 106 mitochondrial absence or presence of SU3327 pretreatment (Fig. 3E). proteins were phosphorylated in the CCl4-exposed samples (Ta- We also evaluated the effect of SU3327 on the levels of lipid ble 1). As summarized in Fig. 5, many proteins, involved in major peroxides as determined by [MDAþHAE]. CCl4 treatment elevated mitochondrial functions such as energy supply, electron transfer, the levels of [MDAþHAE] (Fig. 4A, second lane) compared to those fatty acid oxidation, anti-oxidant defense, chaperones, of vehicle control (first lane) when measured at 24 h, based on the metabolism, etc, were likely phosphorylated by active p-JNK fol- time-dependent increment of lipid peroxides (Fig. 1C). SU3327 lowing CCl4-injection. To systematically evaluate the role of these pretreatment significantly decreased the levels of lipid peroxides mitochondrial proteins in any organ toxicities, we performed (third lane). We also investigated the effects of the mitochondria- molecular toxicity and pathology enrichment analysis of these targeted antioxidant mito-TEMPO [23] on the levels of activated proteins by using MetaCore software. Our analysis revealed that p-JNK and phosphorylated mitochondrial proteins. Consistently many of these phosphoproteins are highly associated with liver with the Fig. 2 results, the levels of active p-JNK and mitochondrial toxicity (Supplemental Fig. S3). phosphoproteins (second lanes of Fig. 4B and C, respectively) were promptly elevated in WT mice exposed to CCl4 for 2 h, compared 3.5. JNK-mediated phosphorylation and activity changes of the se- to vehicle controls (first lanes). Pretreatment with mito-TEMPO lected mitochondrial proteins markedly suppressed the levels of p-JNK and phosphoproteins (third lanes of Fig. 4B and C, respectively), compared to those of To further evaluate the causal roles of phosphoproteins in he- the mice exposed to CCl4 alone (middle lanes in Fig. 4B and C, patotoxicity, we determined the phosphorylation status and ac- respectively). These results suggest the important role of p-JNK tivity changes in the three randomly-selected proteins ALDH2, activated at earlier time points in promoting liver injury observed NdufS1 (a 75-kDa subunit of mitochondrial complex I), and α- at later times through protein phosphorylation. KGDH in control and CCl4-exposed mice in the absence or pre- sence of SU3327 pretreatment. We performed immunoprecipita- 3.4. Identification of mitochondrial phosphoproteins tion by using the specific antibody against each target protein. The immunoprecipitated proteins were then subjected to immunoblot Based on these results with the presence of phosphoproteins in analysis with anti-p-Ser-Pro antibody or antibody against each control mice (Figs. 1–4), we purified phosphorylated mitochon- protein. Immunoblot results showed that the mitochondrial levels drial proteins respectively from vehicle-control and WT mice ex- of ALDH2 remained same regardless of SU3327 pretreatment posed to CCl4 for 2 h by using a metal-affinity phosphoprotein (Fig. 6A, top panel). ALDH2, un-phosphorylated in the untreated purification kit (Qiagen). Subtractive phosphoproteomic analysis control (middle panel, first lane), was phosphorylated in of the mass spectral data with a ratio of CCl4/control groups CCl4-exposed mice (second lane), despite the similar levels of 558 S. Jang et al. / Redox Biology 6 (2015) 552–564

Table 1 Summary of phosphorylated mitochondrial proteins in control and CCl4-exposed mouse livers identified by mass-spectral analysis. Subtraction of phosphoproteins in control (CTRL) and CCl4-exposed (EXP) mice was performed as described in the Section 2.3 of Materials and Methods. Only the proteins with the total area ratios greater than 1.5- fold between EXP and CTRL groups are shown.

Accession Name Description Fold (EXP/ Normalized Intensity_CTRL Normalized Intensity_EXP CTRL) (ppm) (ppm)

IPI00169916.11 Cltc Clathrin heavy chain 1 137.64 1.11Eþ01 1.53Eþ03 IPI00757372.2 Isoc2a Isochorismatase domain-containing protein 2A, 49.97 1.50Eþ01 7.48E þ02 mitochondrial IPI00120212.1 Ndufa9 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex 30.5 1.19Eþ01 3.64Eþ02 subunit 9, mito IPI00319652.2 Gpx1 Glutathione peroxidase 1 30.37 8.33Eþ01 2.53Eþ03 IPI00454049.4 Echs1 Enoyl-CoA hydratase, mitochondrial 28.25 2.38Eþ02 6.72Eþ03 IPI00468653.4 Pccb Propionyl-CoA carboxylase beta chain, mitochondrial 21.86 6.24Eþ01 1.36Eþ03 IPI00153317.3 Aldh1l1 10-formyltetrahydrofolate dehydrogenase 20.48 3.19Eþ02 6.52Eþ03 IPI00122048.2 Atp1a3 Sodium/potassium-transporting ATPase subunit alpha-3 20.16 1.06Eþ02 2.14Eþ03 IPI00330523.1 Pcca Propionyl-CoA carboxylase alpha chain, mitochondrial 18.75 1.42Eþ02 2.66Eþ03 IPI00133553.1 Mut MethyMethylmalonyl-CoA , involved in the de- 18.7 3.40Eþ01 6.36Eþ02 gradation of several amino acids IPI00311682.5 Atp1a1 Sodium/potassium-transporting ATPase subunit alpha-1 15.7 3.95Eþ02 6.21Eþ03 IPI00153660.4 Dlat Acetyltransferase component of pyruvate dehydrogenase 13.88 1.67Eþ02 2.31Eþ03 complex IPI00471246.2 Ivd Isovaleryl-CoA dehydrogenase, mitochondrial 12.41 1.38Eþ01 1.71Eþ02 IPI00420706.4 Lrpprc Leucine-rich PPR motif-containing protein, mitochondrial 11.07 5.64Eþ02 6.24Eþ03 IPI00122549.1 Vdac1 Isoform Pl-VDAC1 of Voltage-dependent anion-selective 10.55 1.87Eþ01 1.97Eþ02 channel protein 1 IPI00114209.1 Glud1 Glutamate dehydrogenase 1, mitochondrial 9.37 3.57Eþ03 3.34Eþ04 IPI00115302.3 Bckdhb Isoform 2 of 2-oxoisovalerate dehydrogenase subunit beta, 9.32 1.38Eþ02 1.29Eþ03 mitochondrial IPI00321718.4 Phb2 Prohibitin-2 9.25 6.36Eþ01 5.88Eþ02 IPI00313998.1 Sqrdl Sulfide:quinone oxidoreductase, mitochondrial 9.12 7.06Eþ02 6.44Eþ03 IPI00308882.4 Ndufs1 NADH-ubiquinone oxidoreductase 75 kDa subunit, 9.04 4.34Eþ02 3.92Eþ03 mitochondrial IPI00331564.2 Dld Dihydrolipoyl dehydrogenase 8.88 5.84Eþ01 5.18Eþ02 IPI00121440.4 Etfb Electron transfer flavoprotein subunit beta 8.6 2.91Eþ02 2.50Eþ03 IPI00420882.3 Ogdh Isoform 4 of 2-oxoglutarate dehydrogenase, mitochondrial 8.36 8.06Eþ00 6.73Eþ01 IPI00453499.3 Iars2 Isoleucyl-tRNA synthetase, mitochondrial 8.2 4.77Eþ01 3.91Eþ02 IPI00130081.2 Pex5 Isoform 2 of Peroxisomal targeting signal 1 receptor 7.42 3.76Eþ01 2.79Eþ02 IPI00230138.7 Lyn Isoform LYN B of Tyrosine-protein kinase Lyn 7.39 1.39Eþ02 1.03Eþ03 IPI00469380.3 Aox3 Aldehyde oxidase 1 7.29 2.94Eþ02 2.14Eþ03 IPI00331322.3 Mgst1 Microsomal glutathione S- 1 7.06 1.73Eþ02 1.22Eþ03 IPI00132799.4 C1qbp complement component 1 Q subcomponent-binding protein, 6.46 1.42Eþ02 9.17Eþ02 mitochondrial IPI00121788.1 Prdx1 Peroxiredoxin 5.91 1.67Eþ02 9.90Eþ02 IPI00676071.3 Mosc1 MOSC domain-containing protein 1, mitochondrial 5.77 1.84Eþ02 1.06Eþ03 IPI00111908.8 Cps1 Carbamoyl-phosphate synthase [ammonia], mitochondrial 5.63 2.39Eþ04 1.34Eþ05 IPI00122862.4 Mthfd1 C-1-tetrahydrofolate synthase, cytoplasmic 5.37 7.54Eþ02 4.05Eþ03 IPI00117312.1 Got2 Aspartate aminotransferase, mitochondrial 5.28 1.18Eþ02 6.23Eþ02 IPI00126625.1 Acsm1 Isoform 1 of Acyl-coenzyme A synthetase ACSM1, 5.11 2.77Eþ02 1.41Eþ03 mitochondrial IPI00109293.1 Lactb Serine beta-lactamase-like protein LACTB, mitochondrial 5.01 7.79Eþ02 3.90Eþ03 IPI00135651.1 Slc25a13 Calcium-binding mitochondrial carrier protein Aralar2 4.73 5.38Eþ02 2.55Eþ03 IPI00312174.6 Ptges2 Microsomal prostaglandin E synthase 2 4.71 7.45Eþ02 3.50Eþ03 IPI00380320.4 Ldhd Probable D-lactate dehydrogenase, mitochondrial 4.53 9.83Eþ01 4.45Eþ02 IPI00116753.4 Etfa Electron transfer flavoprotein subunit alpha, mitochondrial 4.31 8.15Eþ02 3.51Eþ03 IPI00137194.1 Slc16a1 Monocarboxylate transporter 1 4.26 8.95Eþ01 3.81Eþ02 IPI00115117.1 Stoml2 -like protein 2 4.12 5.16Eþ01 2.12Eþ02 IPI00170363.1 Acsl5 Long-chain-fatty-acid-CoA 5 3.89 6.02Eþ02 2.34Eþ03 IPI00319518.4 Lonp1 Lon protease homolog 3.57 1.04Eþ03 3.72Eþ03 IPI00115824.1 Nipsnap1 Protein NipSnap homolog 1 3.3 3.86Eþ02 1.27Eþ03 IPI00117214.3 Hsdl2 Hydroxysteroid dehydrogenase-like protein 2 3.23 2.22Eþ02 7.15Eþ02 IPI00229078.5 Hsd3b4 3-beta-hydroxysteroid dehydrogenase type 4 3.19 2.81Eþ02 8.96Eþ02 IPI00120233.1 Gcdh Glutaryl-CoA dehydrogenase, mitochondrial 2.98 2.61Eþ02 7.78Eþ02 IPI00128286.1 Cyp1a1 Cytochrome P450 1A1 2.95 3.17Eþ02 9.34Eþ02 IPI00320850.3 Mccc1 Methylcrotonoyl-CoA carboxylase subunit alpha 2.91 4.45Eþ02 1.30Eþ03 mitochondrial IPI00122633.3 Acsf2 Acyl-CoA synthetase family member 2, mitochondrial 2.88 9.05Eþ02 2.61Eþ03 IPI00114710.3 Pcx Pyruvate carboxylase, mitochondrial 2.84 3.57Eþ03 1.01Eþ04 IPI00133903.1 Hspa9 Heat shock 70 kDa protein 9 2.8 7.43Eþ03 2.08Eþ04 IPI00136655.1 Gcat 2-amino-3-ketobutyrate coenzyme A ligase, mitochondrial 2.78 3.48Eþ02 9.68Eþ02 IPI00756386.1 Dhtkd1 2-oxoglutarate dehydrogenase E1 component, mitochondrial 2.78 6.06Eþ02 1.69Eþ03 IPI00330754.1 Bdh1 3-hydroxybutyrate dehydrogenase 2.71 2.44Eþ03 6.60Eþ03 IPI00621548.2 Por NADPH-cytochrome P450 reductase 2.7 1.46Eþ03 3.95Eþ03 IPI00136213.5 Sardh Sarcosine dehydrogenase, mitochondrial 2.68 7.96Eþ01 2.14Eþ02 IPI00379694.4 Hmgcl hydroxymethylglutaryl-CoA , mitochondrial precursor 2.55 1.44Eþ02 3.67Eþ02 IPI00308885.6 Hspd1 Isoform 1 of 60 kDa heat shock protein, mitochondrial 2.54 3.47Eþ03 8.81Eþ03 IPI00139301.3 Krt5 Keratin, type II cytoskeletal 5 2.48 8.53Eþ01 2.12Eþ02 IPI00461964.3 Aldh6a1 Methylmalonate-semialdehyde dehydrogenase, 2.48 2.23Eþ03 5.53Eþ03 S. Jang et al. / Redox Biology 6 (2015) 552–564 559

Table 1 (continued )

Accession Name Description Fold (EXP/ Normalized Intensity_CTRL Normalized Intensity_EXP CTRL) (ppm) (ppm)

mitochondrial IPI00111218.1 Aldh2 Aldehyde dehydrogenase, mitochondrial 2.38 2.30Eþ03 5.49Eþ03 IPI00223092.5 Hadha Trifunctional enzyme subunit alpha, mitochondrial 2.37 1.72Eþ03 4.06Eþ03 IPI00270326.1 Psmc2 26S protease regulatory subunit 7 2.37 3.80Eþ01 8.99Eþ01 IPI00226140.5 Maob Amine oxidase [flavin-containing] B 2.36 7.37Eþ02 1.74Eþ03 IPI00331436.4 Lap3 Isoform 1 of Cytosol aminopeptidase 2.29 5.99Eþ01 1.37E þ02 IPI00119114.2 Acadl Long-chain specific acyl-CoA dehydrogenase, mitochondrial 2.26 8.94Eþ02 2.02Eþ03 IPI00116603.1 Otc Ornithine carbamoyltransferase, mitochondrial 2.23 6.57Eþ02 1.47Eþ03 IPI00132042.1 Pdhb Pyruvate dehydrogenase E1-beta subunit, mitochondrial 2.23 1.32Eþ03 2.96Eþ03 IPI00113052.1 Tsfm Elongation factor Ts, mitochondrial 2.22 1.09Eþ02 2.41Eþ02 IPI00118384.1 Ywhae 14-3-3 protein epsilon 2.21 7.79Eþ02 1.72Eþ03 IPI00331555.2 Bckdha 2-oxoisovalerate dehydrogenase subunit alpha, 2.17 8.44Eþ03 1.83Eþ04 mitochondrial IPI00322610.5 Coasy Bifunctional coenzyme A synthase 2.15 1.11Eþ02 2.39Eþ02 IPI00132762.1 Trap1 Heat shock protein 75 kDa, mitochondrial 2.14 2.70Eþ03 5.77Eþ03 IPI00322760.7 Prodh Proline dehydrogenase, mitochondrial 2.14 2.35Eþ03 5.03Eþ03 IPI00112549.1 Acsl1 Long-chain specific acyl-CoA synthetase 1 2.09 4.20Eþ03 8.78Eþ03 IPI00323357.3 Hspa8 Heat shock cognate 71 kDa protein 2.07 2.55Eþ03 5.27Eþ03 IPI00134746.5 Ass1 Argininosuccinate synthase 2.02 1.47Eþ03 2.96Eþ03 IPI00121105.2 Hadh Hydroxyacyl-coenzyme A dehydrogenase, mitochondrial 2.01 1.53Eþ02 3.06Eþ02 IPI00131445.2 Dnm2 Isoform 1 of Dynamin-2 GTPase 1.95 1.89Eþ02 3.70Eþ02 IPI00678532.3 Fam82a2 Regulator of microtubule dynamics protein 3 1.92 1.68Eþ02 3.22Eþ02 IPI00130535.1 Dbt Lipoamide acyltransferase of branched-chain alpha-keto acid 1.9 1.53Eþ03 2.90Eþ03 dehydrogenase complex IPI00130804.1 Ech1 Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase, mitochondrial 1.88 3.75Eþ02 7.07Eþ02 IPI00119203.4 Acadvl Very long-chain specific acyl-CoA dehydrogenase, 1.87 3.41Eþ03 6.36Eþ03 mitochondrial IPI00122075.1 Mavs Mitochondrial antiviral-signaling protein 1.86 4.93Eþ02 9.15Eþ02 IPI00134809.2 Dlst Succinyltransferase component of 2-oxoglutarate dehy- 1.85 4.85Eþ02 8.97Eþ02 drogenase complex IPI00119808.1 Clpx ATP-dependent ClpX-like protease, mitochondrial 1.84 1.06Eþ03 1.95Eþ03 IPI00169862.1 Coq9 Ubiquinone biosynthesis protein COQ9, mitochondrial 1.78 9.68Eþ02 1.73Eþ03 IPI00127841.3 Slc25a5 ADP/ATP 2 1.77 7.18Eþ02 1.27Eþ03 IPI00131177.1 Letm1 LETM1 and EF-hand domain-containing protein 1, 1.75 1.26Eþ03 2.20Eþ03 mitochondrial IPI00408961.3 Haao 3-hydroxyanthranilate 3,4-dioxygenase 1.73 1.06Eþ02 1.83Eþ02 IPI00387491.1 Aass Alpha-aminoadipic semialdehyde synthase, mitochondrial 1.7 5.45Eþ02 9.27Eþ02 IPI00120123.1 Dmgdh Dimethylglycine dehydrogenase, mitochondrial 1.7 1.07Eþ03 1.82Eþ03 IPI00459487.3 Suclg2 Isoform 1 of Succinyl-CoA ligase subunit beta, mitochondrial 1.69 1.61Eþ03 2.72Eþ03 IPI00135231.2 Idh1 Isocitrate dehydrogenase 1.66 5.24Eþ02 8.70Eþ02 IPI00317074.3 Slc25a10 Mitochondrial dicarboxylate carrier 1.61 1.89Eþ02 3.05Eþ02 IPI00115564.5 Slc25a4 ADP/ATP translocase 1 1.6 7.06Eþ02 1.13Eþ03 IPI00116498.1 Ywhaz 14-3-3 protein zeta/delta 1.58 6.25Eþ02 9.89Eþ02 IPI00127625.1 Hmgcl Hydroxymethylglutaryl-CoA lyase, mitochondrial 1.56 8.03Eþ02 1.25Eþ03 IPI00113886.1 Lmnb2 Lamin B2 isoform 1.55 1.13Eþ03 1.76Eþ03 IPI00110684.1 Ppa1 inorganic pyrophosphatase 1.55 3.81Eþ01 5.92Eþ01 IPI00130280.1 Atp5a1 ATP synthase subunit alpha, mitochondrial 1.53 1.51Eþ04 2.32Eþ04 IPI00230108.6 Pdia3 Protein disulfide-isomerase A3 1.53 4.26Eþ03 6.52Eþ03 IPI00113869.1 Bsg Isoform 2 of Basigin 1.53 1.47Eþ02 2.24Eþ02 IPI00119842.1 Acadsb Short/branched chain acyl-CoA dehydrogenase, 1.53 8.58Eþ01 1.32Eþ02 mitochondrial immunoprecipitated ALDH2 protein for each lane (bottom panel). Similar patterns of reversible phosphorylation (Fig. 6E) and

However, the intensity of phosphorylated ALDH2 was markedly activity change (Fig. 6F) in α-KGDH were observed in CCl4-exposed reduced upon SU3327 pretreatment (middle panel, third lane), mice in the absence or presence of SU3327. These results (Fig. 6) suggesting reversible phosphorylation of ALDH2, depending on not only substantiate the presence of phosphoproteins determined the JNK activity. Consistently, the ALDH2 activity in the mi- by mass spectrometry but also support that JNK-mediated phos- tochondrial extracts was significantly suppressed in WT mice ex- phorylation of certain mitochondrial proteins is responsible for the posed to CCl4 for 2 h compared to the control group, but its sup- suppressed activities at 2 h, contributing to CCl4-induced mi- pressed activity was restored in SU3327-pretreated mice (Fig. 6B). tochondrial dysfunction and liver injury observed at later time In addition, the levels of NdufS1 were similar in differently- points (e.g., at 24 h as shown in Fig. 1). treated mice regardless of SU3327 pretreatment (Fig. 6C, top pa- Activated p-JNK can phosphorylate pro-apoptotic Bax to pro- nel). NdufS1 phosphorylation was markedly increased in mote mitochondrial permeability transition (MPT) change and cell

CCl4-exposed WT mice (middle panel, second lane), compared to damage [15,38]. Our results showed that time-dependent mi- the untreated control (first lane). The intensity of the phos- tochondrial translocation of Bax and subsequent release of cyto- phorylated protein band was markedly reduced in SU3327-pre- chrome C were maximal at 2 h post-CCl4 injection (Fig. 7A). treated mice (third lane). Consistently, the mitochondrial complex However, these events were significantly blocked by the SU3327

I activity was significantly decreased in CCl4-exposed WT mice treatment (Fig. 7B). Consistently, CCl4 exposure significantly in- compared with those of control (Fig. 6D). Furthermore, the de- creased time-dependent mitochondrial swelling (Fig. 7C), which creased activity was restored in the SU3327-pretreated group. was significantly prevented by SU3327 pretreatment (Fig. 7D), 560 S. Jang et al. / Redox Biology 6 (2015) 552–564

JNK-mediated phosphorylation of mitochondrial proteins at 2 h after CCl4-exposure

Chaperones and stress response proteins Hsp68, Hsp60, Hsp75, Hsp71 Oxidative phosphorylation Antioxidant defense NADH-ubiquinone oxidoreductase (I) ATP synthase alpha ALDH2 ALDH6a1 Formyltetrahydrofolate dehydrogenase Energy supply Glutathione peroxidase 1 Pyruvate dehydrogenase E1b Pyruvate dehydrogenase E3 Fatty acid β-oxidation Glutamate dehydrogenase Long & short-chain acyl-CoA 2-Oxoisovalerate dehydrogenase alpha dehydrogenase α-Ketoglutarate dehydrogenase E1 E2 Enoyl-CoA hydratase Isocitrate dehydrogenase 3-Hydroxyacyl-CoA dehydrogenase Isovaleryl-CoA dehydrogenase Trifunctional enzyme alpha (thiolase) Branched chain ketoacid dehydrogenase E1 E2 Carbamoyl-phosphate synthase-1 Glutaryl CoA dehydrogenase Electron transport & channel proteins Pyruvate carboxylase Proline dehydrogenase Electron transport flavoprotein beta subunit Voltage dependent anion channel protein 1

Fig. 5. Summary of phosphorylated mitochondrial proteins in WT mice exposed to CCl4 for 2 h. Various functions of mitochondrial proteins that were phosphorylated in

CCl4-exposed mouse liver and identified by mass spectrometry are summarized. The activities of the three proteins marked in blue and bold characters were suppressed in

CCl4-exposed mice compared to the corresponding vehicle-controls. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) supporting JNK-dependent MPT change and mitochondrial dys- hepatotoxicity [13] could result from: (1) the additional activity of function, prior to hepatotoxicity. SP600125 and its non-specific inhibition of other protein kinases [49–52]; (2) potential compensatory mechanisms between the JNK1 and JNK2 isoforms in the liver; (3) potential toxicity of 4. Discussion SP600125 by itself in some cases [53]; (4) a relatively high dosage

of CCl4 in a different mouse strain used for the previous study [13] Molecular mechanisms of acute liver injury caused by many compared to this current study; and (5) no usage of other selective compounds including FDA-approved drugs such as APAP and tro- JNK inhibitors such as SU3327 and BI-78D3 [20–22]. Furthermore, glitazone, an anti-diabetic agent, have been actively studied be- to our knowledge, the identities and functional roles of mi- cause of severe mitochondrial dysfunction and hepatotoxicity [1– tochondrial phosphoprotein targets of active p-JNK have never 4,39–43]. Many abused substances including cocaine and amphe- been studied systematically. Therefore, in the current study, we tamine derivatives are also known to cause mitochondrial dys- aimed to re-evaluate the role of JNK in promoting liver injury function and acute hepatotoxicity [44,45] especially in people who caused by CCl4, as a model of acute hepatotoxicity, by carefully drink alcohol [44]. From the experimental models, many different studying the time-dependent JNK activation and the levels of its mechanisms have been proposed to explain the acute hepato- mitochondrial phosphoprotein targets, lipid peroxides and liver toxicity. These mechanisms include: production of reactive meta- injury with two newly-developed highly-specific JNK inhibitors bolites and protein adducts, increased oxidative/nitrative stress, SU33327 [20] and BI-78D3 [21,22]. Since APAP can cause liver protein modifications and subsequent mitochondrial dysfunction, injury by promoting protein nitration [54,55] in addition to JNK- lipid peroxidation, inflammation through activation of innate im- mediated protein phosphorylation [12,13], we intentionally chose mune responses, toxic ceramide production, activation of the cell to use CCl4 in this study to re-investigate the roles of JNK activa- death pathways, suppression of the cell proliferation/survival tion and JNK-mediated protein phosphorylation at early time pathway, etc. [45–48]. In general, all these mechanisms work to- points prior to mitochondrial dysfunction and acute liver injury, gether toward tissue injury, as recently discussed [47,48]. based on the relatively selective activation of JNK by CCl4 [17,36]. Activation of JNK alone or in combination with p38K is re- We now provide evidence that p-JNK activated at earlier time sponsible for promoting cell death [10,11]. Other laboratories and points plays a pivotal role in promoting CCl4-induced mitochon- we reported the critical role of JNK activation in drug-induced drial dysfunction and hepatotoxicity through phosphorylation of acute hepatotoxicity [12–15,37–39] and ischemia-reperfusion in- many mitochondrial proteins and inactivation of their functions. jury [16]. Although JNK was the main MAPK activated by CCl4 However, these events were markedly reduced in the presence of exposure [17,36] and in our current study (Figs. 1 and 3), another the two new JNK-specific inhibitors SU3327 (Figs. 3, 4 and 6) and report suggested that activated JNK did not seem to be critical in BI-78D3 (not shown). Furthermore, neither JNK activation nor

CCl4-mediated liver injury through evaluation with the ATP-com- hepatotoxicity was observed in CCl4-exposed Cyp2e1-null mice petitive JNK inhibitor SP600125 and the JNK peptide inhibitor-1 despite the same treatment of CCl4 (Fig. 2). We also showed that (D-JNKI-1), which interferes with the interaction between JNK and prompt JNK activation, translocation of active p-JNK to mi- its substrates [13]. The negative role of JNK in CCl4-related tochondria, and protein phosphorylation likely represent the S. Jang et al. / Redox Biology 6 (2015) 552–564 561

Fig. 6. Selected mitochondrial proteins are phosphorylated and their activities suppressed by p-JNK. (A) The level of mitochondrial ALDH2 in control and CCl4-exposed WT mice in the absence or presence of SU3327 pretreatment was determined by immunoblot analysis (top panel). Mitochondrial proteins (0.5 mg/analysis) from vehicle-control and mice exposed to CCl4 for 2 h in the absence or presence of SU3327 were immunoprecipitated with anti-ALDH2 antibody and then subjected to immunoblot analysis with anti-p-Ser-Pro antibody (middle) or anti-ALDH2 antibody (bottom). (B) ALDH2 activity in mitochondrial extracts (0.1 mg protein/assay) for the indicated groups with or without SU3327 pretreatment was determined. Additional analyses were conducted on NdufS1 (complex I) (C, D) and α-KGDH (E, F), as indicated. *, Significantly different (po0.05) from the other groups.

critical events in CCl4-mediated hepatotoxicity than lipid perox- ing mitochondrial complex I, resulting in suppression of the mi- idation [5,6], nitrative stress [54,55], and TNFα-associated in- tochondrial , and thus markedly elevating flammation, as previously suggested [13,35], since these latter oxidative stress, which ultimately contributes to mitochondrial factors were elevated only at 24 h post-CCl4 exposure (Fig. 1 and dysfunction and hepatotoxicity with increased lipid peroxidation data not shown). Based on these time-dependent observations, we observed at later time points. believe that lipid peroxidation and inflammation likely reflect the In addition, JNK was reported to promote cell death through consequences rather than the causes of hepatotoxicity. Further- phosphorylating Bax, which contributes to mitochondrial permeability more, the results with Cyp2e1-null mice (Fig. 2) and mito-TEMPO transition (MPT) change (swelling) and apoptosis [15, 39].Itiswell-

(Fig. 4) indicate that CYP2E1-mediated CCl4 metabolism induces established that activated p-JNK translocates to mitochondria upon oxidative stress, which can rapidly activate JNK (Fig. 2) resulting exposure to various toxic stimulants such as staurosporine, ionizing from inhibition of MAPK phosphatases through oxidative mod- radiation, UV [15] and anisomycin [56].Hanawaetal.reportedthat ifications of their Cys and other critical Cys residues, activated p-JNK was translocated to mitochondria to induce MPT and as recently reviewed [48]. Activated p-JNK then transloc- inhibit mitochondrial respiration, leading to acute hepatocyte injury in ates to mitochondria and phosphorylates many proteins includ- APAP-exposed mice [39]. Our previous report also showed that p-JNK 562 S. Jang et al. / Redox Biology 6 (2015) 552–564

C1 2 4 8 24 kDa Time (h) kDa 23 C Bax 23 C 23 M Bax 23 M

15 C 15 C Cytochrome C Cytochrome C 15 M 15 M C: Cytosol; M: Mitochondria C: Cytosol; M: Mitochondria

2 ## 1.4 # 1.8 1.2 1.6 * 1.4 1 1.2 0.8 1 0.6 0.8 0.6 0.4 0.4 0.2 0.2 0 0 Mitochondria swelling index Mitochondria swelling index Control CCL4 CCL4 + C124824 SU3327 Time (h)

Fig. 7. Time-dependent changes in Bax, cytochrome C, and mitochondrial swelling in CCl4-exposed mice. WT mice were exposed to CCl4 alone for indicated time points (A, C) or only for 2 h in the absence or presence of SU3327 pretreatment (B, D) before tissue collection. (A, B) Time-dependent changes in the cytosolic or mitochondrial amounts of Bax and cytochrome C are presented after determining their levels by immunoblot analysis using the specific antibody to each protein. (C, D) Time-dependent changes in the mitochondrial swelling index for the indicated samples are presented. *,#, Significantly different (po0.05) from the other groups.

was activated in CCl4-exposed rats and translocated to mitochondria activities of phosphorylated mitochondrial proteins despite similar where it could phosphorylate mitochondrial ALDH2 [18]. However, to protein levels (Fig. 6), post-translational modifications of mi- our knowledge, the JNK-target proteins in mitochondria are poorly tochondrial proteins by p-JNK activated at earlier time points understood, although PDH E1α and β subunits, Hsp60 and ATP syn- (Fig. 2) are at least partially responsible for CCl4-induced mi- thase were phosphorylated by active p-JNK in an in vitro system [19]. tochondrial dysfunction prior to hepatotoxicity. Our results Therefore, another aim of this study was to systematically identify showed that α-KGDH and complex I, involved in cell energy sup- mitochondrial target proteins that are phosphorylated by p-JNK and ply, were suppressed in CCl4-exposed tissues compared to control. study their contributing roles in CCl4-related mitochondrial dysfunc- ALDH2, involved in anti-oxidant defense, was also suppressed tion and hepatotoxicity. By using mass-spectral analysis of the affinity- following CCl4 exposure. Since JNK activation and phosphorylation purified phosphoproteins (with more than 2 different peptides iden- occurred earlier (e.g., 1 or 2 h) and led to inactivation of ALDH2, tified by mass spectrometry) and using subtraction phospho-pro- complex I, and α-KGDH, their suppression was likely to promote teomics with the ratio of CCl4/control greater than 1.5, we found that mitochondrial dysfunction, contributing to increased MPT [38], at least 106 mitochondrial proteins, including ALDH2, and eventually liver injury observed at 24 h post-injection. Al- NAD þ -ubiquinone-dehydrogenase, α-KGDH, etc were likely phos- though we have not tested many other phosphoproteins, it is likely phorylated (Table 1). Although our result also confirmed the small list that the activities of some of the phosphorylated mitochondrial of JNK-target phosphoproteins from the in vitro experiment [19],we proteins may be modulated in a JNK-dependent manner. Thus, our believe that the actual number of JNK-target proteins in mitochondria current study provides a novel mechanism for CCl4-induced mi- could be a lot more than what we identified in this study, since the tochondrial dysfunction and hepatotoxicity through JNK-mediated JNK-target proteins existing in small amounts, such as Bax [15] and phosphorylation of many mitochondrial proteins Sab [57,58], may not be detected due to the detection limit of our Although we have purified and identified phosphorylated mi- mass-spectral analysis. Despite this disadvantage, by using im- tochondrial proteins by mass-spectral analysis, we do not know munoprecipitation followed by immunoblot with anti-phospho-Ser- specific phosphorylation sites yet. The canonical JNK binding motif

Pro antibody, we confirmed JNK-mediated phosphorylation of the sequence is: R/K2–3-X1–6-L/I-X-L/I [59]. Based on the comparative three selected target proteins in CCl4-exposed mice. Furthermore, we sequence analysis between mitochondrial ALDH2 and cytosolic demonstrate the functional roles of some of the phosphoproteins since ALDH1, we reported that 463Ser-Pro of ALDH2 could be the prime the suppressed activities of these proteins were restored by JNK in- site of JNK-mediated phosphorylation [18]. We also observed that hibitor SU3327 pretreatment. We expect that the activities of other 75 kDa-subunit NdufS1 subunit of complex I was phosphorylated phosphorylated proteins in CCl4-exposed mice could be also modu- after CCl4 exposure. This protein also contains one putative JNK lated through JNK-mediated phosphorylation. at 515K-R-N-P-P-K-M-L-F-L, and four potential phos- 411 425 588 627 The mechanisms of CCl4-induced mitochondrial dysfunction phorylation sites (Ser -Pro, Ser -Pro, Thr -Pro, Ser -Pro) by have not been fully understood despite previous efforts [5,6 and active p-JNK. Sequence analysis revealed that α-KGDH contains references within]. Based on our results of the suppressed one canonical JNK binding motif at 376K-K-V-M-S-I-L-L, and eight S. Jang et al. / Redox Biology 6 (2015) 552–564 563

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